This invention relates to techniques for the separation and/or purification of biological materials and, more particularly, to a method and apparatus for isoelectric focusing.
Isoelectric focusing ("IEF") also sometimes called electrofocusing, is an electrophoretic technique that is recognized as being a powerful method for the analysis and micropreparative separation and purification of various biological materials, including proteins, peptides, nucleic acids, viruses, and even some living cells or cell organelles. The principle of IEF is based on the fact that certain biomaterials, such as those listed above, are amphoteric in nature, i.e. are positively charged in acidic media and negatively charged in basic media. At a particular pH value, called the isoelectric point, they have a zero net charge. In other words, the isoelectric point is the pH value at which they undergo a reversal of net charge polarity. In a pH gradient such materials will migrate under the influence of a d.c. electric field until they reach the pH of their isoelectric point where they become immobilized by virtue of their zero net charge. Thus, they focus into narrow zones, defined by the pH of the medium and the electric field applied.
IEF techniques have been greatly advanced by the development of suitable buffer systems which form stable pH gradients in the electric field. Such buffers are usually composed of a random mixture of amphoteric substances having isoelectric points covering a wide spectrum of pH values. In the electric field, these components of the buffer mixture are also focused according to their isoelectric points, thereby establishing a stable pH gradient. A commercial mixture of such amphoteric substances called "Ampholine" is available from LKB Produkter AB, a Swedish Company. Other buffer systems are also compatible with IEF. The electric field in IEF thus has two simultaneous and overlapping functions; these being the establishment of the pH gradient and the focusing of the biomaterials to be separated. In terms of time sequence, the establishment of final focusing of the biomaterials cannot be achieved before a stable pH gradient is formed, i.e. before the components of the buffer mixture are focused.
While IEF is widely practiced, it is still limited by the quantities which can be processed and, to applicant's knowledge, IEF is at present used only as an analytical or micropreparative technique. There have been various prior attempts to increase the capacity of IEF. Two recent symposia, where some of the approaches were described, are as follows: (1) P. G. Righetti: Progress in Isoelectric Focusing and Isotachophoresis, North Holland/American Elsevier, 1975 and (2) J. P. Arbuthnott and J. A. Beeley, Isoelectric Focusing, Butterworth, 1975. These volumes also summarize the current status of IEF.
IEF is most often practiced in static, batch-type instruments where the fluid is stabilized by either gels or density gradients established by a non-migrating solute such as sucrose. In such instruments, the capacity for product separation is generally limited by the size of the apparatus to between 1 and 10 mg per cm.sup.2 of apparatus cross-section for each component of the sample applied. Apparatus cross-section cannot be arbitrarily enlarged because of the need to dissipate the Joule heating generated by the electric field. Thus, for larger scale preparative work, it would appear that continuous flow instruments are advantageous. Unfortunately, continuous flow electrophoresis in free solutions is plagued by severe distortions of boundaries of separating materials, caused by several factors: viz., (1) The parabolic nature of liquid flow through confined channels due to viscous drag (flow is fastest through the center of the channel, and decays in a parabolic fashion towards the walls). (2) Electro-osmosis at the walls superimposes another type of parabolic flow, this being in a direction perpendicular to the parabolic profile induced by the viscous drag. (3) Density gradients arising from temperature or sample concentration gradients can cause convective flow of fluid. The disruptive effects of these three factors have been amply described in the literature (cf., for example, K. Hannig et al.: Hoppe-Seyler's Z. Physiol. Chem. Vol. 356, 1209, 1975).
To overcome these difficulties in IEF, two principles of fluid stabilization were tried: stabilization by porous media and stabilization by density gradients (see e.g. J. S. Fawcett, Annals of the New York Academy of Sciences, 209, 112-125, 1973). However, throughput was found to be only comparable to that achievable in static systems. One reason for the limited throughput is that in IEF equilibrium focusing is reached only assymptotically. The rate of electrophoretic migration of each charged species decreases progressively as it approaches its isoelectric point. At the same time, the conductivity of the system decreases as the focused components are less conductive of electricity than when far removed from their isoelectric point. Thus, to obtain sufficient focusing, a relatively long residence time is required, and this is ostensibly achievable either by low flow rates or large apparatus size. A further reason for limited throughput is the dissipation of Joule heat in continuous flow electrophoresis instruments.
It is an object of the present invention to overcome the stated prior art problems and to set forth an IEF technique which exhibits an improved capacity of product separation and purification.